AG126 treatment reduces the myelin lesion size

Representative (immuno-)histological stainings of spinal cord sections are shown in Fig. 4.1B.

Demyelinized white matter lesions were visualized by LFB-PAS-staining. Overall, T cell and microglia/macrophage infiltration of the tissue was visualized by HE, CD 3, or IBA1 staining, respectively. Bielschowsky staining revealed axonal injury. PBS-CTL animals presented with marked lesions of myelinated structures. A pronounced infiltration of immune cells was detected within the lesions as well as a diminished axonal density and looser tissue connections in the affected tissue regions. Tissue of AG126-treated animals showed a reduced damage of the white matter and attenuated lesion characteristics in general. The white matter lesions were quanti-tatively analyzed for consecutive sections, revealing a reduced size in AG126-treated compared to CTL animals (Fig. 4.1C).

Taken together, AG126 reduces the white matter lesions in EAE-induced mice. These data are consistent with the improved disease severity score.

4.1.3. Therapeutic AG126 treatment is more effective than preventive treatment

To determine the ‘kinetics’ of the AG126 effect on the EAE outcome, i.e., the most sensitive period, another EAE study was conducted. Compared to the first one, animals were now treated in two time windows. The first group received the tyrphostin in a preventive manner before the disease onset, starting with day 6 after immunization. The second group was treated in a therapeutic manner. AG126 was applied after disease onset. In contrast to the first EAE experiment, the animals were treated with 500µg AG126/animal/day for five days, i.e., on three consecutive days and thereafter every other day (Fig. 4.2). Moreover, the control group received DMSO as vehicle. Animals were weighted daily and scored according to their disease symptoms (ZTE score). The day after the three consecutive treatments, a blood sample was taken for analysis of immune cells, as described later.

In the preventive AG126 treatment first signs of disease could be detected on day 13 (Fig. 4.2 A). The preventive treatment caused a delay in the course of the disease but could not diminish the extent of extremity paralysis. While in the CTL group, the score maximum was reached on

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Figure 4.2.: AG126 improves the disease course by therapeutic but not preventive treatment in EAE.Wt C57/BL6J mice actively immunized with MOG35−55 were treated on five days (indicated by arrows) with 500µg AG126/animal/day (closed square) or DMSO (control open square). ATreatment started before obvious disease symptoms on day 7 (preventive treatment). B Treatment started after disease onset on day 15 (therapeutic treatment). Animals were weighted and scored daily for clinical signs of the disease on a scale from 0 to 5 (details see Material and Methods section 3.2). Data are mean±SEM, n = 7 animals/group. # Blood samples were taken and analyzed by FACS.

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Figure 4.3.: AG126 shows effects on the CNS but does not affect immune cells in the periphery. Animals were sacrificed on day 30 (compare Fig. 4.2). Histological analysis of spinal cord sections was performed to detect demyelinized white matter (LFB-PAS). AQuantitative analysis of demyelinized spinal cord sec-tions by LFB-PAS staining revealed a tendency for reduced lesion size in AG126-treated animals. Data are mean±SEM (n = 7 per group). B Representative sections with LFB-PAS staining. Asterisks indicate the lesion sites (pink areas within blue stained white matter). C On day 10 (preventive treatment, pv) and day 18 (therapeutic treatment, tp) after MOG immunization, blood samples were taken for the analysis of peripheral blood leukocytes. No effects of AG126 on leukocyte counts in the blood was detectable. FACS analysis was performed for various cell populations (see text). Data are mean±SEM (n = 7 per group).

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4. Results

day 20 (1.2±0.5), the maximum was reached on day 23 (1.4±0.5) in the AG126 group. The maximal mean disease score was even higher than in the DMSO-CTL group.

In the therapeutic treatment, the disease course improved as soon as AG126 administration was started (Fig. 4.2B). No further aggravation (in mean) of clinical symptoms could be observed (1.1±0.4). In contrast, in the DMSO-CTL group, the peak of disease was reached on day 18 (1.7±0.4). Afterwards the disease course constantly declined in the CTL group until day 25 (0.7±0.3). In contrast, the AG126 treated animals showed a relative constant mean disease score until day 27 (0.8±0.3) with little variations between day 16 to 21. In the late phase of the experiment, still some benefit of the AG126 treatment was observed (AG126 0.9±0.4, DMSO-CTL 1.2±0.4). AG126 thus affected the EAE by suppressing the mean disease maxima.

The two treatment regimes came with a slightly different time of onset for the disease symp-toms, i.e., day 11 and 13 for the therapeutic and preventive AG126 administrations. Indeed, animal handling may induce stress, leading to an attenuated disease onset in both treated and control animals (Gold and Heesen, 2006).

Overall, the therapeutic treatment with AG126 suppressed the disease symptoms in com-parison to the DMSO-CTL, whereas preventive treatment just delayed the development of the outcome of the disease symptoms.

Histological analysis of the lesions by LFB-PAS staining revealed reduced white matter damage even in animals with both, preventive or therapeutic treatment (Fig. 4.3A). Representative tissue sections are shown in Fig. 4.3B.

On the other hand, blood analysis for peripheral immune cells, like T cells, total CD11b⁺ cells (covering monocytes, granulocytes, natural killer cells and macrophages), monocyte or granulocyte population revealed no differences between the AG126-treated vs. CTL groups (Fig. 4.3C).

In conclusion, AG126 was able to influence the disease course of animals with an induced EAE. Depending on the time point of AG126 treatment, the tyrphostin attenuated the disease outcome. AG126 thereby represses effectively the disease symptoms, when being applied at onset of clinical signs (compare Fig. 4.1Aand 4.2).

Therefrom, we analyzed the mode of action of AG126 on microglial cells. Here we focused on cellular responses, when cells get activated by TLR ligands, pathogen-associated structures originating from bacteria or viruses.